One major component of our program aimed toward establishing the mechanisms of function of biological metal centers is to explore and expand the contributions that electron nuclear double resonance (endor) spectroscopy can make to metallobiochemistry. To this end we shall develop and apply advanced methodologies, including the use of loop-gap resonators, multi-frequency endor, double endor, and pulsed endor. Using the technique not merely to examine protein resting states, but to probe chemically reactive enzymic intermediates, it will be applied to single site and binuclear copper and iron proteins, peroxidase enzymic intermediates, and complex enzymes, such as cytochrome c oxidase and sulfate reductase, that catalyze the multi-electron reductions of small molecule substrates. The other component is the study of the mechanism of function of hemoproteins, in particular the cooperative binding of ligands to hemoglobin, but also reactive enzymic intermediates of horseradish and cytochrome c peroxidases, and cytochrome P450. Through the use of metal-substituted proteins, employing cobalt, manganese, zinc, and other metalloporphyrins as well, we are able to modify the physical and chemical properties of a protein in significant and interpretable ways, and to introduce new physical and spectroscopic probes with which to observe the influence of a protein environment on a prosthetic group. These metal-substituted proteins, in particular mixed-metal hybrid hemoglobins, also offer unique opportunites in diverse new studies. They will allow direct measurement of long-range (25 Angstroms) electron tunnelling between protein-bound chromosphores that are rigidly held at fixed and crystallographically known distances and orientations. In addition, they will serve as the basis for a new method for examining the gelation of sickle-hemoglobin, and will allow us to characterize the state of free protoporphyrin and zinc protoporphyrin in red blood cells from patients with erythropoietic and iron deficiency anemias, and chronic lead intoxication.